THE USE OF A TRIPLE DETECTION SYSTEM (UV, … be formed, including those found in forced degradation...
1
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2016 Waters Corporation PDA Spectrum (210-400)nm 0.381 - 374.23 0.546 - 432.21 2.958 - 513.27 AU 0.00 0.10 0.20 0.30 QDa Positive Scan CODA SM 5 MCQ 0.75 (QDa Positive(+) Scan (100.00-600.00)Da, Centroid, CV=5) Intensity 0 2x10 6 4x10 6 6x10 6 GlimepirideB XIC - 0.381 - QDa Positive Scan: 374.23 m/z GlimepirideC XIC - 0.546 - QDa Positive Scan: 432.21 m/z Glimepiride XIC - 2.958 - QDa Positive Scan: 513.27 m/z Intensity 0 2x10 6 4x10 6 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Calibration Plot Name: Glimepiride C; Fit Type: Linear (1st Order); R^2: 0.999140; Equation Y = 1.67e+000 X + 2.14e+000 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 LogAmount 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 Calibration Plot Name: Glimepiride C; Fit Type: Quadratic (2nd Order); R^2: 0.996621; Equation Y = 1.62e+001 X^2 + 1.65e+003 X - 2.00e+004 -2.0x10 5 0.0 2.0x10 5 4.0x10 5 6.0x10 5 8.0x10 5 1.0x10 6 1.2x10 6 1.4x10 6 1.6x10 6 Amount 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00 200.00 220.00 240.00 INTRODUCTION In the pharmaceutical industry, there is a need to fully understand the stability of an active pharmaceutical ingredient (API) and characterize any impurities that may be formed, including those found in forced degradation studies. Reversed-phase liquid chromatography UV-based techniques are often used for these types of analyses. However, separating and detecting the related impurities and other components can be challenging. By combining a UV detector, an evaporative light scattering detector, and a mass spectrometer, it is possible to detect compounds with different chemical properties. In the following study, we will conduct a forced degradation study which will also include mass balance studies using triple detection. Mass balance can be affected by the differences in response factors of the API and related impurities. We will perform two sets of experiments to determine the relative response factors (RRF) of the impurities. This process will include either: 1) using the ratio of the slopes of the API and related impurities or 2) comparing the UV peak area to the log of the ELSD peak area, the latter which has a response based on unit mass. Using the relative response factors we will then evaluate the mass balance of the acid hydrolysis of glimepiride. In addition, the QDa mass detector will provide added information for peak confirmation and peak purity. THE USE OF A TRIPLE DETECTION SYSTEM (UV, ELSD, MS) FOR PHARMACEUTICAL DEGRADATION STUDIES Paula Hong , Aaron Phoebe, and Patricia R. McConville Waters Corporation, 34 Maple Street, Milford, MA, USA, 01757 Figure 1. ACQUITY UPLC H-Class system with triple detection including ACQUITY PDA, ELSD and QDa detectors. The triple detection system includes an isocratic solvent manager (ISM) which provides make-up solvent to the QDa detector and houses the splitter required for the ELSD and the QDa. All of the flow is directed to the splitter from the PDA detector. A portion is then sent to the ELSD detector. Make-up solvent is introduced in the next port and the flow is then mixed as it flows into the QDa or mass detector. The composition and flow rate of the make-up solvent impact the split ratio to the ELSD and the QDa. METHODS Conditions System: ACQUITY UPLC H-Class with Column Manager Column: ACQUITY UPLC BEH C18, 1.7 μm, 2.1 x 50 mm Mobile phase A: 0.1% (v/v) Formic acid in Water Mobile phase B: 0.1% (v/v) Formic acid in Acetonitrile Column Temperature: 30 C Injection volume: 2 uL Flow rate: 0.8 mL/min Isocratic:60% A: 40%B Gradient: ACQUITY PDA Detector Wavelength range: 210-400 nm Resolution: 3.6 nm Selected wavelengths: 228 nm, 4.8 nm resolution Time Constant: Normal Sampling rate: 20 pts/s ACQUITY Evaporative Light Scattering Detector (ELSD) Gas: 25 psi Neubilizer Mode: Cooling Nebulizer Temperature: 55 ˚C Gain: 350 Data rate: 10 pps Isocratic Solvent Manager Flow rate: 0.3 mL/min Solvent: 0.1% (v/v) formic acid in methanol ACQUITY QDa Detector (mass detector) Mass range: 100-600 Cone voltage: 5V Sampling Rate: 5 pps Capillary Voltage: 1.4 kV Sample Preparation: Glimepiride and related compounds B and C were purchased from the USP. All standards were dissolved in 55:45 methanol:water and sonicated. The drug substance glimepiride was obtained from an outside source. Acid hydrolysis was conducted at 40 ˚C for 0-7 days . The concentration of acid was 0.1M HCl in the degradation reaction. References 1. Chapter <621> CHROMATOGRAPHY United States Pharmacopeia and National Formulary (USP 37-NF 32 S1) Baltimore, MD: United Book Press, Inc.; 2014. p. 6376-85. 2. Mark AN, Andreas K, Patrick JJ. Role of Mass Balance in pharmaceutical stress testing. Pharmaceutical Stress Testing: CRC Press; 2011. p. 233-53. 3. Bansal G, Singh M, Jindal KC, Singh S. LC–UV–PDA and LC–MS studies to characterize degradation products of glimepiride. Journal of pharmaceutical and biomedical analysis. 2008; 48:788-95. Ensuring Peak Purity using Mass and UV detection CONCLUSIONS Triple detection system in combination with Empower 3 FR2 provides various tools to assist in mass balance, including: Determination of relative response factors by using the ratio of UV peak and the log of ELSD peak responses The ability to input relative response values into Empower 3 FR2 to determine corrected area values for impurities for mass balance determinations Using orthogonal detection (UV and mass) to confirm peak purity and to detect the presence of co-elutions that could impact mass balance. Mass Balance for Forced Degradation Studies Time %A %B %C %D Curve Initial 95.0 5.0 0.0 0.0 Initial 5.00 5.0 95.0 0.0 0.0 6 6.50 5.0 95.0 0.0 0.0 6 6.51 95.0 5.0 0.0 0.0 6 Samples Conditions API Control RT and 40°C for 1,3,5, 7 days Acid control RT and 40°C for 1,3,5, 7 days Acid Hydrolysis RT and 40°C for 1,3,5, 7 days Table 3. Mass balance determinations for forced degradation of glimepiride. The calculations were performed using RRF de- termined with the ELSD method. The RRF were entered into Empower 3 FR 2 for corrected values of the related impurities. All mass balance values were within 2%. AU 0.00 0.20 0.40 374.3 432.2 513.2 AU 0.00 0.20 0.40 AU 0.00 0.10 0.20 0.30 0.40 0.50 Minutes 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 t=0 t= 3 days t= 5 days Figure 7. UV chromatograms of forced degradation of glimepiride drug substance with base mass labels. The drug substance was exposed to acidic hydrolysis conditions at 40 C over a period of days. Over the course of the study the two impurity peaks ( related compound C and B) increased in peak area. Figure 2. Forced degradation of glimepiride drug substance. AU 0.00 0.05 0.10 0.15 Minutes 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 LSU 0.00 10.00 20.00 Minutes 1.00 2.00 3.00 4.00 5.00 Intensity 0.0 5.0x10 5 1.0x10 6 1.5x10 6 2.0x10 6 Minutes 1.00 2.00 3.00 4.00 5.00 PDA @ 228 nm ELSD MS XIC ESI+ Figure 3. Separation of standards of active pharmaceutical ingredient (API), related compound B and related compound C under isocratic conditions. The overlay of standards at 250 μg/mL for the API and 10 μg/mL for related compounds B and C shows the differences in relative response among the detectors. The UV and ELSD give similar relative response for the three compounds. In the mass detector related compound C has a greater peak area than related compound B. Figure 5. Overlay of glimepiride related compound C standards (10-250 μg/mL) in PDA and ELSD. The UV detector produces a linear response for standards. Evaluating the peak areas in the ELSD, a non-linear or logarithmic response is observed. For example, at 10 μg/mL the response in the ELSD (pink trace) is significantly lower than that observed in the PDA. Wavelength Separation Conditions RRF Rel Compound B RRF Rel Compound C 228 Isocratic 1.36 1.10 Name: GlimepirideB; Fit Type: Linear (1st Order); R^2: 0.999945; Equation Y = 1.06e+004 X - 1.78e+001 Name: Glimepiride; Fit Type: Linear (1st Order); R^2: 0.999469; Equation Y = 7.72e+003 X - 1.56e+003 Name: Glimepiride C; Fit Type: Linear (1st Order); R^2: 0.998663; Equation Y = 8.86e+003 X - 1.76e+003 -4.0x10 5 -2.0x10 5 0.0 2.0x10 5 4.0x10 5 6.0x10 5 8.0x10 5 1.0x10 6 1.2x10 6 1.4x10 6 1.6x10 6 1.8x10 6 2.0x10 6 2.2x10 6 ppm 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 Area -2.0x10 5 0.0 2.0x10 5 4.0x10 5 6.0x10 5 8.0x10 5 1.0x10 6 1.2x10 6 ppm 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 Figure 4. Overlay of UV linearity curves at 228nm of glimepiride and related impurities B and C. The API linearity curve covers the range of 1-250 μg/mL for the API and 1-50 μg/mL for related compounds B and C. All R 2 values were >0.998. AU 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 Minutes 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 PDA @ 228 nm 250 μg/mL 125 μg/mL 50 μg/mL 10 μg/mL LSU 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 Minutes 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 250 μg/mL 125 μg/mL 50 μg/mL 10 μg/mL ELSD Figure 6. ELSD calibration curves for glimepiride related compound C. The ELS detector has a quadratic fit to the calibration curve (top) for peak area vs. the amount. If the values are converted to the logarithmic functions (inset), the calibration curve fit is linear (bottom). The R 2 value for this curve is 0.999140. Standard RRF Rel Compound B RRF Rel Compound C 50 1.25 1.10 125 1.24 1.09 ELSD calibration curve Quadratic fit ELSD calibration curve Linear fit Table 1. Relative Response Factors for Related compound B and C using the ratio of the slope of the API/slope of the impurity. The value for related compound B is outside of 0.8-1.2 range and, therefore, should be applied, as specified by the USP Chapter <621>. 1 RESULTS AND DISCUSSION Multi-detection of API and Related Compounds Determination of Relative Response Factors Using Slope of Calibration Curves in UV = [ ] [ ] Determination of Relative Response Factors Using Ratio of UV to ELSD (log) Peak Area Y-axis X-axis LogArea LogAmount Table 2. Relative Response Factors for related compound B and C using the ratio of the UV peak area to the log of the ELSD peak area. RRF can be calculated using the response of the UV detector to a mass concentration dependent detector. 2 This assumes a linear relationship for both detectors. To convert the ELSD calibration response to a linear function, the log of both x and y values can be used. Thereby, using the log of the ESLD peak area, we can calculate RRF factors for both impurities. These values have good correlation with those obtained using the slopes of the calibration curves in the UV. = log ( / = / n = 3 Reference T=0 1 day 3 days 5 days 7 days Amount 238.4 236.1 239.0 242.7 239.1 244.1 % Recovery 99.0 100.2 101.8 100.3 102.4 Figure 8. Mass analysis window in Empower 3 FR2 of forced degradation at 3 days. In forced degradation studies, mass imbalance can be caused by co-elutions of the API and any impurities. To evaluate peak purity, mass detection can provide valuable information. In this view, the UV, Total Ion Chromatogram (TIC) and Extracted Ion Chromatograms (XIC) are shown in a stacked plot. For each integrated peak in the UV chromatogram the leading, apex and trailing portion of the peak can be viewed (top portion of figure). Using the mass and UV detectors the peak purity can be evaluated to ensure no co- elutions. Pre-configured Splitter in the Isocratic Solvent Manager ACQUITY UPLC H-Class System with Column Managers and Triple Detection (UV-ELSD-PDA) API Rel Cmpd B Rel Cmpd C API Rel Cmpd B Rel Cmpd C GlimepirideC - 0.546 - QDa Positive Scan Leading Apex Trailing 228.3 294.5337.2359.0 228.3 228.3 300.1 316.7355.2 432.26 432.21 432.30 UV TIC, ESI+ XIC, ESI+ Peak purity view of glimepiride related compound C + HCl Δ Flow diagram
Transcript of THE USE OF A TRIPLE DETECTION SYSTEM (UV, … be formed, including those found in forced degradation...
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2016 Waters Corporation
Project Name: Mass Balance Degrad Nov15Reported by User: System
Report Method: Untitled Date Printed:
125 12/1/2015Report Method ID: 125
11:17:57 AM US/Eastern
GlimepirideB - 0.381 - QDa Positive Scan
Leading Apex Trailing
222.8
300.1339.0381.3
226.5 226.5
298.2 360.8
374.05
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514.96
374.23 374.12
374.72
432.44
GlimepirideC - 0.546 - QDa Positive Scan
Leading Apex Trailing
228.3
294.5337.2359.0
228.3 228.3
300.1316.7355.2
432.26 432.21 432.30
PDA Spectrum (210-400)nm
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.23
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.27
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QDa Positive Scan CODA SM 5 MCQ 0.75 (QDa Positive(+) Scan (100.00-600.00)Da, Centroid, CV=5)
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GlimepirideB XIC - 0.381 - QDa Positive Scan: 374.23 m/z
GlimepirideC XIC - 0.546 - QDa Positive Scan: 432.21 m/z
Glimepiride XIC - 2.958 - QDa Positive Scan: 513.27 m/z
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Project Name: MassBalance Glimep 14Oct15Reported by User: System
Report Method: Calibration curve ELSD stack Date Printed:
24851 2/26/2016Report Method ID: 24851
9:49:19 AM US/Eastern
Calibration Plot
Name: Glimepiride C; Fit Type: Linear (1st Order); R^2: 0.999140; Equation Y = 1.67e+000 X + 2.14e+000
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Glimepiride C
Glimepiride C
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Glimepiride C
Glimepiride C
Glimepiride C
Glimepiride C
Glimepiride C
1.000000
1.000000
1.000000
1.698970
1.698970
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2.096910
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2.397940
3.838315
3.805043
3.820003
4.921575
4.942371
4.964010
5.629349
5.648075
5.678314
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1.018281
0.998353
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1.667098
1.679553
1.692515
2.091018
2.102234
2.120345
2.386219
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0.731
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-1.143
-0.380
-0.281
0.254
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Peak: Glimepiride C
Calibration curve ELSD stack
Project Name: MassBalance Glimep 14Oct15Reported by User: System
Report Method: Calibration curve ELSD stack Date Printed:
24851 2/26/2016Report Method ID: 24851
9:50:31 AM US/Eastern
Calibration Plot
Name: Glimepiride C; Fit Type: Quadratic (2nd Order); R^2: 0.996621; Equation Y = 1.62e+001 X̂ 2 + 1.65e+003 X - 2.00e+004
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Point Information ' Peak: GlimepirideB' table contains no data.
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Name Level X Value Response Calc. Value % Deviation Manual Ignore
Glimepiride C
Glimepiride C
Glimepiride C
Glimepiride C
Glimepiride C
Glimepiride C
Glimepiride C
Glimepiride C
Glimepiride C
Glimepiride C
10.000000
10.000000
10.000000
50.000000
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50.000000
125.000000
125.000000
125.000000
250.000000
6891.517641
6383.265905
6606.982471
83478.507006
87573.059729
92047.180381
425941.030337
444707.880031
476775.136351
1324998.044518
14.265562
14.025072
14.131038
43.764007
45.086203
46.510456
122.439136
125.740811
131.244015
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Peak: Glimepiride C
INTRODUCTION
In the pharmaceutical industry, there is a need to fully
understand the stability of an active pharmaceutical ingredient (API) and characterize any impurities that
may be formed, including those found in forced degradation studies. Reversed-phase liquid
chromatography UV-based techniques are often used for these types of analyses. However, separating and
detecting the related impurities and other components can be challenging. By combining a UV detector, an
evaporative light scattering detector, and a mass
spectrometer, it is possible to detect compounds with different chemical properties.
In the following study, we will conduct a forced
degradation study which will also include mass balance studies using triple detection. Mass balance can be
affected by the differences in response factors of the API and related impurities. We will perform two sets of
experiments to determine the relative response factors (RRF) of the impurities. This process will include either:
1) using the ratio of the slopes of the API and related
impurities or 2) comparing the UV peak area to the log of the ELSD peak area, the latter which has a response
based on unit mass. Using the relative response factors we will then evaluate the mass balance of the acid
hydrolysis of glimepiride. In addition, the QDa mass detector will provide added information for peak
confirmation and peak purity.
THE USE OF A TRIPLE DETECTION SYSTEM (UV, ELSD, MS) FOR PHARMACEUTICAL DEGRADATION STUDIES
Paula Hong, Aaron Phoebe, and Patricia R. McConville
Waters Corporation, 34 Maple Street, Milford, MA, USA, 01757
Figure 1. ACQUITY UPLC H-Class system with triple detection
including ACQUITY PDA, ELSD and QDa detectors. The triple
detection system includes an isocratic solvent manager (ISM)
which provides make-up solvent to the QDa detector and houses
the splitter required for the ELSD and the QDa. All of the flow is
directed to the splitter from the PDA detector. A portion is then
sent to the ELSD detector. Make-up solvent is introduced in the
next port and the flow is then mixed as it flows into the QDa or
mass detector. The composition and flow rate of the make-up
solvent impact the split ratio to the ELSD and the QDa.
METHODS
Conditions
System: ACQUITY UPLC H-Class with Column Manager
Column: ACQUITY UPLC BEH C18, 1.7 µm, 2.1 x 50 mm
Mobile phase A: 0.1% (v/v) Formic acid in Water
Mobile phase B: 0.1% (v/v) Formic acid in Acetonitrile
Column Temperature: 30 C
Injection volume: 2 uL
Flow rate: 0.8 mL/min
Isocratic:60% A: 40%B
Gradient:
ACQUITY PDA Detector
Wavelength range: 210-400 nm
Resolution: 3.6 nm
Selected wavelengths: 228 nm, 4.8 nm resolution
Time Constant: Normal
Sampling rate: 20 pts/s
ACQUITY Evaporative Light Scattering Detector (ELSD)
Gas: 25 psi
Neubilizer Mode: Cooling
Nebulizer Temperature: 55 ˚C
Gain: 350
Data rate: 10 pps
Isocratic Solvent Manager
Flow rate: 0.3 mL/min
Solvent: 0.1% (v/v) formic acid in methanol
ACQUITY QDa Detector (mass detector)
Mass range: 100-600
Cone voltage: 5V
Sampling Rate: 5 pps
Capillary Voltage: 1.4 kV
Sample Preparation:
Glimepiride and related compounds B and C were purchased from the USP.
All standards were dissolved in 55:45 methanol:water and sonicated.
The drug substance glimepiride was obtained from an outside source. Acid
hydrolysis was conducted at 40 ˚C for 0-7 days . The concentration of acid
was 0.1M HCl in the degradation reaction.
References
1. Chapter <621> CHROMATOGRAPHY United States Pharmacopeia and National Formulary (USP 37-NF 32
S1) Baltimore, MD: United Book Press, Inc.; 2014. p. 6376-85.
2. Mark AN, Andreas K, Patrick JJ. Role of Mass Balance in pharmaceutical stress testing. Pharmaceutical
Stress Testing: CRC Press; 2011. p. 233-53.
3. Bansal G, Singh M, Jindal KC, Singh S. LC–UV–PDA and LC–MS studies to characterize degradation
products of glimepiride. Journal of pharmaceutical and biomedical analysis. 2008; 48:788-95.
Ensuring Peak Purity using Mass and UV detection
CONCLUSIONS
Triple detection system in combination with Empower
3 FR2 provides various tools to assist in mass
balance, including:
Determination of relative response factors by using
the ratio of UV peak and the log of ELSD peak responses
The ability to input relative response values into
Empower 3 FR2 to determine corrected area values for impurities for mass balance determinations
Using orthogonal detection (UV and mass) to
confirm peak purity and to detect the presence of
co-elutions that could impact mass balance.
Mass Balance for Forced Degradation Studies
Time %A %B %C %D Curve
Initial 95.0 5.0 0.0 0.0 Initial
5.00 5.0 95.0 0.0 0.0 6
6.50 5.0 95.0 0.0 0.0 6
6.51 95.0 5.0 0.0 0.0 6
Samples Conditions
API Control RT and 40°C for 1,3,5, 7 days
Acid control RT and 40°C for 1,3,5, 7 days
Acid Hydrolysis RT and 40°C for 1,3,5, 7 days
Table 3. Mass balance determinations for forced degradation
of glimepiride. The calculations were performed using RRF de-termined with the ELSD method. The RRF were entered into
Empower 3 FR 2 for corrected values of the related impurities. All mass balance values were within 2%.
AU
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Figure 7. UV chromatograms of forced degradation of glimepiride
drug substance with base mass labels. The drug substance was exposed to acidic hydrolysis conditions at 40 C over a period of
days. Over the course of the study the two impurity peaks ( related compound C and B) increased in peak area.
Figure 2. Forced degradation of glimepiride drug substance.
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PDA @ 228 nm
ELSD
MS XIC
ESI+
Figure 3. Separation of standards of active pharmaceutical ingredient (API), related compound B and related compound C under
isocratic conditions. The overlay of standards at 250 µg/mL for the
API and 10 µg/mL for related compounds B and C shows the
differences in relative response among the detectors. The UV and ELSD give similar relative response for the three compounds. In the mass detector related compound C has a greater peak area than
related compound B.
Figure 5. Overlay of glimepiride related compound C standards
(10-250 µg/mL) in PDA and ELSD. The UV detector produces a
linear response for standards. Evaluating the peak areas in the
ELSD, a non-linear or logarithmic response is observed. For
example, at 10 µg/mL the response in the ELSD (pink trace) is
significantly lower than that observed in the PDA.
Wavelength
Separation
Conditions
RRF Rel
Compound B
RRF Rel
Compound C
228 Isocratic 1.36 1.10
Calibration curve
Project Name: MassBalance Glimep 14Oct15Reported by User: System
Report Method: Calibration curve Date Printed:
18823 12/2/2015Report Method ID: 18823
1:42:05 PM US/Eastern
Calibration Plot
Name: GlimepirideB; Fit Type: Linear (1st Order); R^2: 0.999945; Equation Y = 1.06e+004 X - 1.78e+001
Name: Glimepiride; Fit Type: Linear (1st Order); R^2: 0.999469; Equation Y = 7.72e+003 X - 1.56e+003
Name: Glimepiride C; Fit Type: Linear (1st Order); R^2: 0.998663; Equation Y = 8.86e+003 X - 1.76e+003
Are
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50.000000
50.000000
10427.014939
10322.367898
10382.729387
53820.208641
54051.671831
53601.392108
106798.664119
107004.997494
107492.919871
529969.685196
529309.806651
528377.102249
0.983969
0.974110
0.979797
5.071877
5.093682
5.051263
10.062776
10.082214
10.128179
49.928110
49.865945
49.778079
-1.603
-2.589
-2.020
1.438
1.874
1.025
0.628
0.822
1.282
-0.144
-0.268
-0.444
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Peak: GlimepirideB
Calibration curve
Project Name: MassBalance Glimep 14Oct15Reported by User: System
Report Method: Calibration curve Date Printed:
18823 12/2/2015Report Method ID: 18823
1:45:01 PM US/Eastern
Calibration Plot
Name: GlimepirideB; Fit Type: Linear (1st Order); R^2: 0.999945; Equation Y = 1.06e+004 X - 1.78e+001
Name: Glimepiride; Fit Type: Linear (1st Order); R^2: 0.999469; Equation Y = 7.72e+003 X - 1.56e+003
Name: Glimepiride C; Fit Type: Linear (1st Order); R^2: 0.998663; Equation Y = 8.86e+003 X - 1.76e+003
Are
a
-2.0x105
0.0
2.0x105
4.0x105
6.0x105
8.0x105
1.0x106
1.2x106
ppm
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00
1
2
3
4
5
6
7
8
9
10
11
12
Name Level X Value Response Calc. Value % Deviation Manual Ignore
GlimepirideB
GlimepirideB
GlimepirideB
GlimepirideB
GlimepirideB
GlimepirideB
GlimepirideB
GlimepirideB
GlimepirideB
GlimepirideB
GlimepirideB
GlimepirideB
1.000000
1.000000
1.000000
5.000000
5.000000
5.000000
10.000000
10.000000
10.000000
50.000000
50.000000
50.000000
10427.014939
10322.367898
10382.729387
53820.208641
54051.671831
53601.392108
106798.664119
107004.997494
107492.919871
529969.685196
529309.806651
528377.102249
0.983969
0.974110
0.979797
5.071877
5.093682
5.051263
10.062776
10.082214
10.128179
49.928110
49.865945
49.778079
-1.603
-2.589
-2.020
1.438
1.874
1.025
0.628
0.822
1.282
-0.144
-0.268
-0.444
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Peak: GlimepirideB
Figure 4. Overlay of UV linearity curves at 228nm of glimepiride
and related impurities B and C. The API linearity curve covers
the range of 1-250 µg/mL for the API and 1-50 µg/mL for
related compounds B and C. All R2 values were >0.998.
AU
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
Minutes
0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
PDA @
228 nm
250 µg/mL 125 µg/mL 50 µg/mL 10 µg/mL
LS
U
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
800.00
900.00
Minutes
0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
250 µg/mL 125 µg/mL 50 µg/mL 10 µg/mL
ELSD
Figure 6. ELSD calibration curves for glimepiride related
compound C. The ELS detector has a quadratic fit to the
calibration curve (top) for peak area vs. the amount. If the
values are converted to the logarithmic functions (inset), the
calibration curve fit is linear (bottom). The R2 value for this
curve is 0.999140.
Standard
RRF Rel Compound B
RRF Rel Compound C
50 1.25 1.10
125 1.24 1.09
ELSD calibration curve
Quadratic fit
ELSD calibration curve Linear fit
Table 1. Relative Response Factors for Related compound B and
C using the ratio of the slope of the API/slope of the impurity.
The value for related compound B is outside of 0.8-1.2 range
and, therefore, should be applied, as specified by the USP
Chapter <621>.1
RESULTS AND DISCUSSION
Multi-detection of API and Related Compounds
Determination of Relative Response Factors Using Slope
of Calibration Curves in UV
𝑅𝑅𝐹 = [𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝐼𝑚𝑝𝑢𝑟𝑖𝑡𝑦]
[𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝐴𝑃𝐼]
Determination of Relative Response Factors Using Ratio of UV to ELSD (log) Peak Area
Calibration curve
Project Name: MassBalance Glimep 14Oct15Reported by User: System
Report Method: Calibration curve Date Printed:
23805 12/28/2015Report Method ID: 23805
8:36:43 PM US/Eastern
Calibration Plot
Name: Glimepiride; Fit Type: Linear (1st Order); R^2: 0.996948; Equation Y = 1.84e+000 X + 1.38e+000
Name: GlimepirideB; Fit Type: Linear (1st Order); R^2: 0.999125; Equation Y = 1.69e+000 X + 2.25e+000
Name: GlimepirideB; Fit Type: Linear (1st Order); R^2: 0.999125; Equation Y = 1.69e+000 X + 2.25e+000
Name: Glimepiride; Fit Type: Linear (1st Order); R^2: 0.996948; Equation Y = 1.84e+000 X + 1.38e+000
Name: Glimepiride C; Fit Type: Linear (1st Order); R^2: 0.999140; Equation Y = 1.67e+000 X + 2.14e+000
Name: Glimepiride; Fit Type: Linear (1st Order); R^2: 0.996948; Equation Y = 1.84e+000 X + 1.38e+000
Lo
gA
rea
1.00
2.00
3.00
4.00
5.00
6.00
7.00
ppm
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40
1
2
3
4
5
Name Y-axis X-axis Fit Type R^2 Equation
GlimepirideB
Glimepiride C
Glimepiride
Glimepiride C
Glimepiride
LogArea
LogArea
LogArea
LogAmount
LogAmount
LogAmount
Linear (1st Order)
Linear (1st Order)
Linear (1st Order)
0.999125
0.999140
0.996948
Y = 1.69e+000 X + 2.25e+000
Y = 1.67e+000 X + 2.14e+000
Y = 1.84e+000 X + 1.38e+000
Table 2. Relative Response Factors for related compound B and
C using the ratio of the UV peak area to the log of the ELSD
peak area. RRF can be calculated using the response of the UV
detector to a mass concentration dependent detector.2 This
assumes a linear relationship for both detectors. To convert the
ELSD calibration response to a linear function, the log of both x
and y values can be used. Thereby, using the log of the ESLD
peak area, we can calculate RRF factors for both impurities.
These values have good correlation with those obtained using
the slopes of the calibration curves in the UV.
𝑅𝑅𝐹 = 𝑈𝑉 𝐴𝑟𝑒𝑎𝐼𝑚𝑝𝑢𝑟𝑖𝑡𝑦
𝐸𝐿𝑆𝐷 log(𝐴𝑟𝑒𝑎 𝑖𝑚𝑝𝑢𝑟𝑖𝑡𝑦/
𝑈𝑉 𝐴𝑟𝑒𝑎𝐴𝑃𝐼𝐸𝐿𝑆𝐷 𝐴𝑟𝑒𝑎 𝐴𝑃𝐼
𝑅𝑅𝐹 = 𝑈𝑉 𝐴𝑟𝑒𝑎𝐼𝑚𝑝𝑢𝑟𝑖𝑡𝑦
𝐶𝑜𝑛𝑐 𝐷𝑒𝑡𝑒𝑐𝑡𝑜𝑟 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑖𝑚𝑝𝑢𝑟𝑖𝑡𝑦/
𝑈𝑉 𝐴𝑟𝑒𝑎𝐴𝑃𝐼𝐶𝑜𝑛𝑐 𝐷𝑒𝑡𝑒𝑐𝑡𝑜𝑟 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝐴𝑃𝐼
n = 3
Reference
T=0
1 day
3 days
5 days
7 days
Amount
238.4 236.1 239.0 242.7 239.1 244.1
% Recovery
99.0 100.2 101.8 100.3 102.4
Figure 8. Mass analysis window in Empower 3 FR2 of forced
degradation at 3 days. In forced degradation studies, mass
imbalance can be caused by co-elutions of the API and any
impurities. To evaluate peak purity, mass detection can provide
valuable information. In this view, the UV, Total Ion
Chromatogram (TIC) and Extracted Ion Chromatograms (XIC)
are shown in a stacked plot. For each integrated peak in the UV
chromatogram the leading, apex and trailing portion of the peak
can be viewed (top portion of figure). Using the mass and UV
detectors the peak purity can be evaluated to ensure no co-
elutions.
Pre-configured Splitter in the
Isocratic Solvent Manager
ACQUITY UPLC H-Class System
with Column Managers and Triple
Detection (UV-ELSD-PDA)
API Rel Cmpd B
Rel Cmpd C
API
Rel Cmpd B
Rel Cmpd C
Project Name: Mass Balance Degrad Nov15Reported by User: System
Report Method: Untitled Date Printed:
125 12/1/2015Report Method ID: 125
11:17:57 AM US/Eastern
GlimepirideB - 0.381 - QDa Positive Scan
Leading Apex Trailing
222.8
300.1339.0381.3
226.5 226.5
298.2 360.8
374.05
491.19
514.96
374.23 374.12
374.72
432.44
GlimepirideC - 0.546 - QDa Positive Scan
Leading Apex Trailing
228.3
294.5337.2359.0
228.3 228.3
300.1316.7355.2
432.26 432.21 432.30
PDA Spectrum (210-400)nm
0.3
81
- 3
74
.23
0.5
46
- 4
32
.21
2.9
58
- 5
13
.27
AU
0.00
0.10
0.20
0.30
QDa Positive Scan CODA SM 5 MCQ 0.75 (QDa Positive(+) Scan (100.00-600.00)Da, Centroid, CV=5)
Inte
ns
ity
0
2x106
4x106
6x106
GlimepirideB XIC - 0.381 - QDa Positive Scan: 374.23 m/z
GlimepirideC XIC - 0.546 - QDa Positive Scan: 432.21 m/z
Glimepiride XIC - 2.958 - QDa Positive Scan: 513.27 m/z
Inte
ns
ity
0
2x106
4x106
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00
UV
TIC, ESI+
XIC, ESI+
Peak purity view of glimepiride related
compound C
+ HCl Δ
Flow diagram
